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General Recommendations and Guidelines for PCR

PCR is a powerful technique capable of amplifying trace amounts of DNA. All appropriate precautions should be taken to avoid cross-contamination.

MgSO4: MgSO4 is included in the 10X AccuPrime™ Pfx Reaction Mix at a final concentration of 1 mM, which is sufficient for most templates. For further optimization, add 0.1 μl to 1.0 μl of 50-mM MgSO4 (included in the kit) to the reaction.

dNTPs: dNTPs are included in the 10X AccuPrime™ Pfx Reaction Mix at a final concentration of 0.3 mM.

Annealing Temperature: The optimal annealing temperature should be 5–10°C lower than the Tm of the primers used; if necessary, gradually increase the annealing temperature by 2–3°C for higher specificity.

KCl: For difficult primer sets, prepare titrations of KCl (not included) at final concentrations of 20–50 mM for further optimization.

PCR Protocol

The following general procedure is suggested as a starting point when using AccuPrime™ Pfx DNA Polymerase in any PCR amplification.

Add the following components to an autoclaved microcentrifuge tube at either room temperature or on ice:

Component

Volume

Final Concentration

10X AccuPrime™ Pfx Reaction mix*

5 μl

1X

Primer mix (10 μM each)*

1.5 μl

0.3 μM each

Template DNA (10 pg–200 ng)

≥ 1 μl

as required

AccuPrime™ Pfx DNA Polymerase**

0.4–1 μl

1.0–2.5 units

Autoclaved distilled water

to 50 μl

*AccuPrime™ Pfx DNA Polymerase will not function in reactions that contain dUTP either in the primers or in the dNTP mix.
**For most targets, 1 unit is optimal. Higher concentrations may be inhibitory. More enzyme may be required for longer targets (>3 kb).

Mix contents of the tubes and overlay with mineral or silicone oil, if necessary. (Note: The oil overlay is unnecessary in thermal cyclers equipped with a heated lid.)

Cap the tubes and centrifuge briefly to collect the contents.

Denature the template for 2 min at 95°C. Perform 25–35 cycles of PCR amplification as follows: